Targeting Aggressive B-cell Lymphomas through Pharmacological Activation of the Mitochondrial Protease OMA1.

DLBCL are aggressive, rapidly proliferating tumors that critically depend on the ATF4-mediated integrated stress response (ISR) to adapt to stress caused by uncontrolled growth, such as hypoxia, amino acid deprivation, and accumulation of misfolded proteins. Here, we show that ISR hyperactivation is a targetable liability in DLBCL. We describe a novel class of compounds represented by BTM-3528 and BTM-3566, which activate the ISR through the mitochondrial protease OMA1. Treatment of tumor cells with compound leads to OMA1-dependent cleavage of DELE1 and OPA1, mitochondrial fragmentation, activation of the eIF2α-kinase HRI, cell growth arrest, and apoptosis. Activation of OMA1 by BTM-3528 and BTM-3566 is mechanistically distinct from inhibitors of mitochondrial electron transport, as the compounds induce OMA1 activity in the absence of acute changes in respiration. We further identify the mitochondrial protein FAM210B as a negative regulator of BTM-3528 and BTM-3566 activity. Overexpression of FAM210B prevents both OMA1 activation and apoptosis. Notably, FAM210B expression is nearly absent in healthy germinal center B-lymphocytes and in derived B-cell malignancies, revealing a fundamental molecular vulnerability which is targeted by BTM compounds. Both compounds induce rapid apoptosis across diverse DLBCL lines derived from activated B-cell, germinal center B-cell, and MYC-rearranged lymphomas. Once-daily oral dosing of BTM-3566 resulted in complete regression of xenografted human DLBCL SU-DHL-10 cells and complete regression in 6 of 9 DLBCL patient-derived xenografts. BTM-3566 represents a first-of-its kind approach of selectively hyperactivating the mitochondrial ISR for treating DLBCL.

Permissive ureter specification by TBX18-mediated repression of metanephric gene expression.

The murine kidney and ureter develop in a regionalized fashion from the ureteric bud and its surrounding mesenchyme. Whereas the factors that establish the metanephric cell lineages have been well characterized, much less is known about the molecular cues that specify the ureter. Here, we have identified a crucial patterning function in this process for Tbx18, a T-box transcription factor gene specifically expressed in the mesenchymal primordium of the ureter. Using misexpression and loss-of-function mice combined with molecular profiling approaches, we show that Tbx18 is required and sufficient to repress metanephric mesenchymal gene programs. We identify Wt1 as a functional target of TBX18. Our work suggests that TBX18 acts as a permissive factor in ureter specification by generating a mesenchymal domain around the distal ureteric bud where SHH and BMP4 signaling can occur.

Real-Time Characterization of Clonal Fate Decisions in Complex Leukemia Samples by Fluorescent Genetic Barcoding.

Clonal heterogeneity in acute myeloid leukemia (AML) forms the basis for treatment failure and relapse. Attempts to decipher clonal evolution and clonal competition primarily depend on deep sequencing approaches. However, this prevents the experimental confirmation of the identified disease-relevant traits on the same cell material. Here, we describe the development and application of a complex fluorescent genetic barcoding (cFGB) lentiviral vector system for the labeling and subsequent multiplex tracking of up to 48 viable AML clones by flow cytometry. This approach allowed the visualization of longitudinal changes in the in vitro growth behavior of multiplexed color-coded AML clones for up to 137 days. Functional studies of flow cytometry-enriched clones documented their stably inherited increase in competitiveness, despite the absence of growth-promoting mutations in exome sequencing data. Transplantation of aliquots of a color-coded AML cell mix into mice revealed the initial engraftment of similar clones and their subsequent differential distribution in the animals over time. Targeted RNA-sequencing of paired pre-malignant and de novo expanded clones linked gene sets associated with Myc-targets, embryonic stem cells, and RAS signaling to the foundation of clonal expansion. These results demonstrate the potency of cFGB-mediated clonal tracking for the deconvolution of verifiable driver-mechanisms underlying clonal selection in leukemia.

Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells.

DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34+ stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.

Proteomic analysis identifies ZMYM2 as endogenous binding partner of TBX18 protein in 293 and A549 cells.

The TBX18 transcription factor regulates patterning and differentiation programs in the primordia of many organs yet the molecular complexes in which TBX18 resides to exert its crucial transcriptional function in these embryonic contexts have remained elusive. Here, we used 293 and A549 cells as an accessible cell source to search for endogenous protein interaction partners of TBX18 by an unbiased proteomic approach. We tagged endogenous TBX18 by CRISPR/Cas9 targeted genome editing with a triple FLAG peptide, and identified by anti-FLAG affinity purification and subsequent LC-MS analysis the ZMYM2 protein to be statistically enriched together with TBX18 in both 293 and A549 nuclear extracts. Using a variety of assays, we confirmed the binding of TBX18 to ZMYM2, a component of the CoREST transcriptional corepressor complex. Tbx18 is coexpressed with Zmym2 in the mesenchymal compartment of the developing ureter of the mouse, and mutations in TBX18 and in ZMYM2 were recently linked to congenital anomalies in the kidney and urinary tract (CAKUT) in line with a possible in vivo relevance of TBX18-ZMYM2 protein interaction in ureter development.

A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML.

HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.

Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes.

Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung.

BACKGROUND: Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. METHODS: We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. RESULTS: We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. CONCLUSION: Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.

Delayed onset of smooth muscle cell differentiation leads to hydroureter formation in mice with conditional loss of the zinc finger transcription factor gene Gata2 in the ureteric mesenchyme.

The establishment of the peristaltic machinery of the ureter is precisely controlled to cope with the onset of urine production in the fetal kidney. Retinoic acid (RA) has been identified as a signal that maintains the mesenchymal progenitors of the contractile smooth muscle cells (SMCs), while WNTs, SHH, and BMP4 induce their differentiation. How the activity of the underlying signalling pathways is controlled in time, space, and quantity to activate coordinately the SMC programme is poorly understood. Here, we provide evidence that the Zn-finger transcription factor GATA2 is involved in this crosstalk. In mice, Gata2 is expressed in the undifferentiated ureteric mesenchyme under control of RA signalling. Conditional deletion of Gata2 by a Tbx18cre driver results in hydroureter formation at birth, associated with a loss of differentiated SMCs. Analysis at earlier stages and in explant cultures revealed that SMC differentiation is not abrogated but delayed and that dilated ureters can partially regain peristaltic activity when relieved of urine pressure. Molecular analysis identified increased RA signalling as one factor contributing to the delay in SMC differentiation, possibly caused by reduced direct transcriptional activation of Cyp26a1, which encodes an RA-degrading enzyme. Our study identified GATA2 as a feedback inhibitor of RA signalling important for precise onset of ureteric SMC differentiation, and suggests that in a subset of cases of human congenital ureter dilatations, temporary relief of urine pressure may ameliorate the differentiation status of the SMC coat. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mesothelial mobilization in the developing lung and heart differs in timing, quantity, and pathway dependency.

The mesothelial lining of the lung, the visceral pleura, and of the heart, the epicardium, derive from a common multipotent precursor tissue, the mesothelium of the embryonic thoracic cavity that also contributes to organ-specific mesenchymal cell types. Insight into mesothelial mobilization and differentiation has prevailedin the developing heart while the mesenchymal transition and fate of the visceral pleura are poorly understood. Here, we use the fact that the early mesothelium of both the lung and the heart expresses the transcription factor gene Wt1, to comparatively analyze mesothelial mobilization in the two organs by a genetic cre-loxP-based conditional approach. We show that epicardial cells are mobilized in a large number between E12.5 and E14.5, whereas pleural mobilization occurs only sporadically and variably in few regions of the lung in a temporally highly confined manner shortly after E12.5. Mesothelium-specific inactivation of unique pathway components using a Wt1creERT2 line excluded a requirement for canonical WNT, NOTCH, HH, TGFB, PDGFRA, and FGFR1/FGFR2 signaling in the mesenchymal transition of the visceral pleura but indicated a deleterious effect of activated WNT, NOTCH, and HH signaling on lung development. Epicardial mobilization was negatively impacted on by loss of HH, PDGFRA, FGFR1/2 signaling. Epicardial overactivation of WNT, NOTCH, and HH disturbed epicardial and myocardial integrity. We conclude that mesothelial mobilization in the developing lung and heart differs in timing, quantity and pathway dependency, indicating the organ specificity of the program.

Proteomic analysis identifies transcriptional cofactors and homeobox transcription factors as TBX18 binding proteins.

The TBX18 transcription factor is a crucial developmental regulator of several organ systems in mice, and loss of its transcriptional repression activity causes dilative nephropathies in humans. The molecular complexes with which TBX18 regulates transcription are poorly understood prompting us to use an unbiased proteomic approach to search for protein interaction partners. Using overexpressed dual tagged TBX18 as bait, we identified by tandem purification and subsequent LC-MS analysis TBX18 binding proteins in 293 cells. Clustering of functional annotations of the identified proteins revealed a highly significant enrichment of transcriptional cofactors and homeobox transcription factors. Using nuclear recruitment assays as well as GST pull-downs, we validated CBFB, GAR1, IKZF2, NCOA5, SBNO2 and CHD7 binding to the T-box of TBX18 in vitro. From these transcriptional cofactors, CBFB, CHD7 and IKZF2 enhanced the transcriptional repression of TBX18, while NCOA5 and SBNO2 dose-dependently relieved it. All tested homeobox transcription factors interacted with the T-box of TBX18 in pull-down assays, with members of the Pbx and Prrx subfamilies showing coexpression with Tbx18 in the developing ureter of the mouse. In summary, we identified and characterized new TBX18 binding partners that may influence the transcriptional activity of TBX18 in vivo.

A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

BMP4 uses several different effector pathways to regulate proliferation and differentiation in the epithelial and mesenchymal tissue compartments of the developing mouse ureter.

Heterozygous loss of Bmp4 results both in humans and mice in severe malformation of the urinary tract. These defects have at least partially been attributed to loss of expression of Bmp4 in the ureteric mesenchyme, yet the cellular and molecular function of this signal as well as its effector pathways in this tissue have remained incompletely resolved. Here, we show that mice with a conditional deletion of Bmp4 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional and physical ureter obstruction. Proliferation in both the mesenchymal and epithelial progenitor pools was severely reduced and smooth muscle cell and urothelial differentiation programs were not activated. Epithelial expression of P-ERK1/2, P-AKT and P-P38, and mesenchymal expression of P-SMAD1/5/9, P-P38 and P-AKT were abrogated. Pharmacological inhibition and activation experiments in ureter cultures defined AKT as the most relevant downstream effector for epithelial and mesenchymal proliferation as well as for epithelial differentiation. Epithelial proliferation and differentiation were also influenced by P-38 and ERK1/2, while SMAD signaling, together with AKT and P-38, were required for smooth muscle cell differentiation. Our analysis suggests that BMP4 is the signal that couples the proliferation and differentiation programs in the epithelial and mesenchymal tissue compartments of the developing ureter by different downstream effectors, most importantly AKT and SMAD.

Tbx2 and Tbx3 Act Downstream of Shh to Maintain Canonical Wnt Signaling during Branching Morphogenesis of the Murine Lung.

Numerous signals drive the proliferative expansion of the distal endoderm and the underlying mesenchyme during lung branching morphogenesis, but little is known about how these signals are integrated. Here, we show by analysis of conditional double mutants that the two T-box transcription factor genes Tbx2 and Tbx3 act together in the lung mesenchyme to maintain branching morphogenesis. Expression of both genes depends on epithelially derived Shh signaling, with additional modulation by Bmp, Wnt, and Tgfß signaling. Genetic rescue experiments reveal that Tbx2 and Tbx3 function downstream of Shh to maintain pro-proliferative mesenchymal Wnt signaling, in part by direct repression of the Wnt antagonists Frzb and Shisa3. In combination with our previous finding that Tbx2 and Tbx3 repress the cell-cycle inhibitors Cdkn1a and Cdkn1b, we conclude that Tbx2 and Tbx3 maintain proliferation of the lung mesenchyme by way of at least two molecular mechanisms: regulating cell-cycle regulation and integrating the activity of multiple signaling pathways.

Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT.

Hypoxia and the anticoagulants dalteparin and acetylsalicylic acid affect human placental amino acid transport.

BACKGROUND: Anticoagulants, e.g. low-molecular weight heparins (LMWHs) and acetylsalicylic acid (ASA) are prescribed to women at risk for pregnancy complications that are associated with impaired placentation and placental hypoxia. Beyond their role as anticoagulants these compounds exhibit direct effects on trophoblast but their impact on placental function is unknown. The amino acid transport systems A and L, which preferably transfer essential amino acids, are well-described models to study placental nutrient transport. We aimed to examine the effect of hypoxia, LMWHs and ASA on the activity of the placental amino acid transport systems A and L and associated signalling mechanisms. METHODS: The uptake of C14-MeAIB (system A) or H3-leucin (system L) was investigated after incubation of primary villous fragments isolated from term placentas. Villous tissue was incubated at 2% O2 (hypoxia), 8% O2 and standard culture conditions (21% O2) or at 2% O2 and 21% O2 with dalteparin or ASA. Activation of the JAK/STAT or mTOR signalling pathways was determined by Western analysis of total and phosphorylated STAT3 or Raptor. RESULTS: Hypoxia decreased system A mediated MeAIB uptake and increased system L mediated leucine uptake compared to standard culture conditions (21% O2). This was accompanied by an impairment of STAT3 and a stimulation of Raptor signalling. System L activity increased at 8% O2. Dalteparin treatment reduced system A and system L activity under normoxic conditions and ASA (1 mM) decreased system A and L transporter activity under normoxic and hypoxic conditions. CONCLUSIONS: Our data underline the dependency of placental function on oxygen supply. LMWHs and ASA are not able to reverse the effects of hypoxia on placental amino acid transport. These findings and the uncovering of the signalling mechanisms in more detail will help to understand the impact of LMWHs and ASA on placental function and fetal growth.